Past work has established that many important cargos move bi-directionally along microtubules, and that the distribution and net transport of such cargos can be controlled by regulating the relative contributions of the plus-end versus minus-end motors. It has also established that multiple plus-end and multiple minus-end motors function together. However, both the fundamental mechanism of regulation of such transport is unclear, as is the importance (and regulation) of the number of engaged motors. The work proposed here will lead to a much deeper understanding both of the regulation of transport, and also of the control and importance of motor number in this regulation. Specifically, bi-directional motion of lipid droplets in early Drosophila embryos is investigated. The work can be conceptually divided into two complimentary approaches. In the first, we develop and critically test a theoretical model for how this transport is controlled by tuning single-molecule properties. The theoretical approach is an extension of our past modeling of transport driven by multiple kinesin motors, and rely on Monte Carlo simulations. The theoretical predictions are then tested using genetics to alter protein dosage of the motors, and also specific point mutants. Some of the testing of the model relies on quantifying-in vivo-the motion of individual cargos with high temporal and spatial resolution, and determining if the motion is consistent with the model's prediction. In the second, we focus on the regulation of the number of engaged motors and their force production, as controlled by the klar protein. We use a variety of biophysical characterizations to determine the effects of klar mutations. In particular, we use optical tweezers and particle tracking and analysis to determine the specific physical role of different domains of the klar protein, and then use complementary biochemical and cell biological techniques to determine the molecular interactions underpinning these physical roles. This information will clarify at the molecular level how the biophysically determined functions come about, and because we have already shown that klar regulates force production (and likely number of engaged motors), the study will directly probe how the number of engaged motors is controlled, and the functional implications of this control. PUBLIC HEALTH RELEVANCE: Bi-directional transport is directly related to public health: viruses such as herpes spread through cells in a bi-directional manner;many important cargos like mitochondria and endosomes move bi-directionally. Impaired vesicular transport is implicated in Neuronal degeneration. Finally, a better understanding of transport might allow the design of new drug delivery systems.